Robinson

Question 1

Purpose of endosomes

Answer 1

Sorting of endocytosed material

Question 2

Protein import into the ER (ER destined)

Answer 2

N-terminal signal sequence, 15-20 AAs long
SRP binds to sequence on growing polypeptide
SRP binds to SRP receptor near translocation channel
SRP leaves, growing chain pushes through channel
Signal sequence left in membrane, cleaved by signal peptidase

Question 3

Structure of SRP

Answer 3

Ribonucleoprotein
7S RNA and various proteins
Binding of signal sequence is modulated by NAC
Elongation is stopped until SRP docks

Question 4

The Sec61 channel structure

Answer 4

Used for import to ER
Hetero trimeric complex of aby subunits
Also used for post translational with Hsp70/Hsp40

Question 5

Embedding proteins into the ER

Answer 5

Sequence of hydrophobic stop transfer

Anchors to membrane

Question 6

Function of the ER

Answer 6

Protein folding- PDI rearranges disulphide bonds. Chaperones can bind to hydrophobic regions
Glycosylation
Quality control

Question 7

Glycosylation of proteins

Answer 7

Lipid linked oligosaccharide
Asn-X-Ser/Thr
Anchored to membrane by -P- to dolichol
Cleaved from membrane to join to Asn by transferase

Question 8

ER quality control-Protein folding

Answer 8

N-glycosylation increases the solubility of unstructured chains. Chaperones and folding factors are recruited when time for folding expired

Misfolded proteins remain bound to chaperones and are degraded

Question 9

ER quality control- Glucose trimming

Answer 9

Terminal glucoses are removed by a-glucosidases to form the Glc1Man9GlcNAc2 sugar
Glucosidase I and II remove 2 outermost residues belonging to N-glycans branch A
Chaperones bind to exposed hydrophobic regions and prevent vesicles

Question 10

Chaperone proteins

Answer 10

Receptors IRE1 and PERK are activated by oligermerisation and trans phosphorylation when misfolded proteins bind
The Bip inhibitor leaves the receptor

IRE1 cleaves XBP1 mRNA to activate it
IRE1 also forms RIDD -> mRNA attached to ribosome degradation

PERK phosphorylates eIF2 to inhibit translation
ATF4 (mRNA) escapes block and creates -> CHOP + GADD34
These are factors for amino acid metabolism

When bip is released, activates ATF6 receptor
ATF6 Trans located to golgi -> luminal domain cleaved
Moves to nucleus and expresses molecules for chaperone pathways

Question 11

Purpose of ER

Answer 11

Lipid synthesis

Secretory pathway

Question 12

Principles of mitochondrial import

Answer 12

Imported post translationally but unfolded
Made with transient n terminal extension
Internal targeting signals not removed
Trans located through TOM
Presequence directed to TIM23
Inner membrane -> TIM22

Question 13

TIM23

Answer 13

Presequence containing proteins
Example of sequence- COXIV sequence is an Amphipathic helix
Transport depends on Ψ and mtHsp70(ATPase)

Question 14

Mitochondrial targeting sequences

Answer 14

Positive hydrophobic residues
Amphipathic helices, used to bind to receptor in outer membrane
Positive charge required for Ψ driven transport
Cleaved matrix proteins have an Arg at -2 or -3
Processing matrix by MPP

Question 15

Import of COXIV-DHFR with methotrexate

Answer 15

Methotrexate- protein only processed by MPP
Must have 50+ residues between folded DHFR and processing site
Tim23 and TOM are stacked at contact sites where 2 membranes are close enough for translocation

Question 16

Structure of TOM complex

Answer 16

Tom 20 -> 22 -> TOM40

Tom80 -> TOM40

Question 17

PAM complex

Answer 17

PAM 1618
Tim44
mtHsp70
Mge1
This acts as an ATP powered pulling mechanism

Question 18

Brownian ratchet model

Answer 18

Protein must be unfolded
Brownian movement only causes fluctuation across membrane
Protein trapped so cannot move back
Dragged through, unwinding

Protein targeting needs ATP
translocation needs ATP and electrical gradient

Question 19

Inner and outer membrane protein structure

Answer 19

Inner are a helical

Out are b barrel

Question 20

Stop transfer mechanism

Answer 20

Proteins arrested in membrane during translocation
E.g. CoxVA in inner membrane
Cleavage by matrix protease
Stop transfer sequence

Question 21

Conservative sorting

Answer 21

Protein is imported into matrix
Instead by ancestral pathway 
Example- ATP synthase unit 9 
Cleavage by matrix protease
Internal sequences recognised by Oxa1

Question 22

What determines the route of an inner membrane protein

Answer 22

Proteins without homologue use stop transfer (TIM proteins)
Proteins with bacterial homologues use conservative sorting
Could be due to sequence and charges around hydrophobic domain

Question 23

TIM22

Answer 23

Similar to TIM23
Import of AAC (an ADP ATP carrier) and phosphate carrier (PiC)
In the preprotein Sequences are recognised by Tom70 and Tim22
Chaperone through tom70 and into general import pore linked to tim9/10
This transports it to tim22/54

Question 24

Targeting signals of chloroplast proteins

Answer 24

Hydroxylated amino acids, 30-100 residues
May be phosphorylated at Ser or Thr
Transit peptides bind targeting factor in cytosol
Assisted by cytosolic chaperones
Recognised by TOC
Presequences removed by stromal processing peptidase
Thylakoid have additional targeting signal

Question 25

Recognition of preprotein by TOC

Answer 25

Preprotein -> GTPase domain of Toc159 Complex docks at outer membrane via interaction with toc159 and toc34 Toc159/34 interaction -> GTP hydrolysis -> integration of toc159 and insertion of preprotein After translocation toc139 and 34 exchanges GDP for GTP Dissociation of toc159

Question 26

Translocation by the TOC complex

Answer 26

Transit peptide binds to toc34 Stimulates GTP hydrolysis by toc34 which transfers preprotein to toc159 GTP hydrolysis by toc159 causes conformational change forcing the preprotein through the translocation channel More rounds of GTP hydrolysis completes process

Question 27

3 stages of chloroplast envelope translocation

Answer 27

Transit peptide makes reversible contacts with receptors of the TOC complex Preprotein inserts into the TOC complex and makes contact with TIC components (low ATP conc) Preprotein is trans located into Stroma and the TP is cleaved by SPP (high ATP needed)

Question 28

Import of proteins into the thylakoid lumen

Answer 28

Made with bipartite targeting signal One for transport across envelope, one for transport into thylakoid Lumenal proteins follow Sec or Tat pathway Depends on signal peptide Transport is post translational

Question 29

Sec pathway

Answer 29

Transported unfolded Depends on ATP and stromal proteins or cytoplasmic proteins SPase removes the targeting signal at the trans side of the membrane After translocation, proteins fold to active conformation

Question 30

Tat pathway

Answer 30

Transported folded Relies solely on proton motive force SPase removes the targeting signal at the trans side of the membrane Conserved residues in targeting signals

Question 31

Protein export in bacteria

Answer 31

Putative RR signal peptide Consensus sequence S/TRRxFLK Tat substrates in e.coli bind cofactors and are involved in respiration Cofactor insertion takes place in the cytoplasm, why folded proteins are trans located

Question 32

Possible mechanism for the tat system

Answer 32

Two seperate complexes are present TatA + tatABC complex Only tatBC in thylakoids Substrate targeted to the membrane and binds to tatBC tatA is then recruited to the core complex to make a pore of the right size The protein is trans located TatA dissociates to close the pore

Question 33

Why transport proteins to the nucleus

Answer 33

Nuclear function proteins are synthesised in the cytoplasm Nuclear envelope breaks down in mitosis Nuclear and cytoplasm mix Components must be resorted after cell division

Question 34

Nuclear localisation signal + gated transport

Answer 34

Pro-pro-Lys-Lys-Lys-Arg This needs to be on surface Recognised by nuclear import receptors These bind to nuclear pore fibrils extending into cytosol Protein and receptor enter nucleus Import receptor recycled Gated transport- larger than 60kDa need ATP

Question 35

Mechanism of nuclear pore transport

Answer 35

Nuclear localisation signal associates with import receptors These bind the nuclear pore complex (act as chaperone) Complex moves through pore (unknown) Once inside, Ran-GTP displaces import receptor Ran-GTP receptor complex travels out of nucleus GTP hydrolysis and receptor released in cytoplasm

Question 36

Monomeric GTPases

Answer 36

Cell has high GTP, so only anti-clockwise GTP -> GDP by GAP removing Pi Hydrolysis energy used to power cellular functions Reverse reactions stimulated by GEF

Question 37

Ran-GAP/GEF in nuclear transport

Answer 37

Ran-GAP -> lots of Ran-GDP in cytosol In the nucleus there is lots of Ran-GEF therefore Ran-GTP High ran-GTP promotes dissociation of NLS from import receptor Most nucleus export is RNA

Question 38

Export of RNA from nucleus

Answer 38

Forms complex of ribonucleoprotein The protein component contains a NES that is recognised by export proteins mRNA is bound by hnRNP after full splicing (ensures mature)

Question 39

Modification of oligosaccharide added in ER

Answer 39

Enzymes in cisternae can add and remove sugar residues | Also phosphorylation of mannose residues

Question 40

Role of protein glycosylation

Answer 40

Inhibition does not affect secretion, may affect folding Oligosaccharides can make protein resistant to protease digestion Also function in cell to cell adhesion Connected via Asn

Question 41

Specific proteolytic cleavage

Answer 41

Some proteins are synthesised as larger precursor proteins and then cleaved by TGN molecules to activate Some proteins entering Golgi need to return to ER, ER retention signal recognised by cis golgi

Question 42

ER -> Golgi transport

Answer 42

Proteins bud off in vesicles and form a vesiculotubular cluster (vesicles with vesicles inside) This travels along micro tubules to the Golgi It then breaks down into ER-Golgi intermediate component Assembly of new cis-Golgi cisternae from VTC elements

Question 43

Protein sorting in the trans Golgi network | 4 destinations

Answer 43

4 destinations- stay, lysosomes, constitutive or regulated secretion

Question 44

Lysosomes and trans Golgi network

Answer 44

Acidic ph due to proton pump Lysosomal enzymes and membrane proteins synthesised in ER Mannose residue is phosphorylated at Golgi Mannose-6-phosphate recognised by vesicle coat proteins Clathrin coat bound to adaptin and directed to lysosomes

Question 45

Sorting receptor for lysosomes

Answer 45

Cargo binds to sorting receptor Sorting receptor binds to adaptin which binds to clathrin to make coat Vesicle buds off to lysosomes

Question 46

Vesicle budding

Answer 46

Clathrin molecules shape vesicle Dynamin assembles round neck of vesicle Hydrolases GTP contracting the ring and vesicle leaves

Question 47

Structure of clathrin

Answer 47

Triskelion shape Each leg has heavy and light chain These polymerise to form a polygonal lattice with an intrinsic curvature

Question 48

COP I coated vesicles (Golgi -> ER)

Answer 48

Requires ARF to release GDP and bind GTP complex then binds to ARF receptors on Golgi COP I coatamers bind to ARF and other proteins inducing budding

Question 49

COP II coated vesicles (ER -> Golgi)

Answer 49

GEF in donor membrane interacts with GTPase Sar1 -> exchange Sar1-GTP extends fatty acid tail that extends into membrane COPII assembles on Sar1 to form vesicle

Question 50

Vesicle fusion

Answer 50

Uncoating exposes vSNARE ,recognised by tSNARE/SNAP25 complex NSF, a/b/gSNAP proteins bind to stabilise energy barrier Rab-GTP hydrolysed, causes fusion and releases complex Vesicle with vSNARES returns to donor membrane

Question 51

Conformational changes in vSNARE tSNARE complex

Answer 51

Stalk formation Hemi fusion Fusion This expels water, lipid contacts involve syntaxin, synaptobrevin and SNAP25

Question 52

Separation of the vSNARE/tSNARE complex

Answer 52

NSF cycles between the membrane and cytosol catalysing disassembly Uses ATP to unravel interactions Stops tSNARES from always being active Ran monitors NSF

Question 53

Constitutive pathway

Answer 53

Bulk flow All proteins except for ER and Golgi retention, transport to lysosomes or secretory vesicles Plasma membrane proteins and lipids etc. Some secretion to blood e.g. Albumin

Question 54

Regulated pathway

Answer 54

Selective aggregation in trans Golgi Secondary structure important Uptake of aggregate into immature secretory vesicles

Question 55

Endocytosis pathways

Answer 55

Pinocytosis- fluid and molecules, all cells | Phagocytosis- large molecules, specialised cells e.g.macrophage

Question 56

Fates for proteins after endocytosis

Answer 56

Recycling- receptors to same place on membrane Transcytosis- different place on membrane Degradation- take to lysosomes

Question 57

Serine proteases

Answer 57

Chymotrypsin- likes aromatic side chain on residue whos carbon is being cleaved Trypsin- prefers a positively charged Lys or Arg residue There is Nucleophillic attack of the hydroxyl O of a Ser on the carbonyl carbon Forms an acyl enzyme intermediate

Question 58

Aspartate proteases

Answer 58

Pepsin, Remin, HIV-protease One Asp accepts a proton from an active site H2O which attack the carbonyl carbon of the peptide linkage The other -/0 donates a proton to the oxygen of the peptide carbonyl group at the same time

Question 59

Zinc proteases

Answer 59

Carboxypeptidases, matrix metalloproteases, one lysosomal protease Some involved in degradation of EM during tissue remodelling (collagen) Some have roles in cell signalling, can release cytokines or growth factors by cleave of membrane preproteins

Question 60

Cystein proteases

Answer 60

Also known as Capthespsins De protonation of the Cys SH by adjacent His residue Followed by Nucleophillic attack of the cysteine S on peptide carbonyl carbon Thioester linking the new carboxyterminus to cysteine thiol is an intermediate of the reaction

Question 61

Protein turnover

Answer 61

N-end rule, half life correlates to N terminal residue Phe, Leu, Asp have less than 3 mins PEST proteins very fast

Question 62

Joining of ubiquitin to protein

Answer 62

Terminal carboxyl of ubiquitin joined to Cys on E1 (ATP dependent) Ubiquitin transferred to a S group of E2 E3 transfers activated ubiquitin to e amino group of Lys of protein recognised by E3 More ubiquitins are added to form chain Linked by Lys residues 4+ targets proteins for degradations

Question 63

Destruction box sequence

Answer 63

Recognised by a domain of the corresponding ubiquitin ligase | Present in mitotic cyclins etc.

Question 64

Ubiquitin ligases (E3)

Answer 64

Some have HECT domain containing a Cys residue that participates in ubiquitin transfer Some have a RING finger where Cys and His residues are ligands to 2Zn2+ ions

Question 65

The proteasome core complex

Answer 65

20S sedimentation coefficient 7 a type proteins form 2 a rings at the ends 7 b type proteins form the 2 central b rings Cavity with 3 joined compartments Proteases activities are associated with 3 of the b subunits

Question 66

The 3 b protease subunits

Answer 66

Chymotrypsin like activity- preference for Tyr or Phe at P1 Trypsin like activity- Arg or Lys at P1 Post-glutamyl activity- Glu or acidic residue at P1 Unique family of threonine proteases, conserved Thr involved at catalysis at each active site. Pre proteins activated by cleavage -> taken to lumenal surface

Question 67

The 19S cap

Answer 67

Unfolds ubiquitin proteins, removes ubiquitn and provides passage Outermost lid is ring of 8 proteins Innermost base is 6 ATPases - unfolding chaperones Isopeptidases disassemble ubiquitin and recycle 19S + 20S -> 26S proteasome

Question 68

The 11S cap

Answer 68

Regulatory cap- heptameric complex of PA28 Allows non ubiquitin proteins and peptides to pass through Binding alters conformation of core complex a units opening gate